This research proposal is focused on the progesterone receptor (PR) and two domains of PR, the amino terminal domain (NTD) and the carboxyl terminal extension (CTE) of the DNA binding domain (DBD). The NTD harbors a transcriptional activation domain (AF-1) that is important for target gene and cell type specific transcriptional activities of PR. The short CTE interacts with coregulatory proteins and binds DNA flanking progesterone response elements (PRE) to extend the protein-DNA interface and stabilize interactions mediated by the core DBD. Despite the functional importance of the NTD and CTE these domains remain poorly defined relative to the ligand binding (LBD) and DBD, in part because they are intrinsically disordered protein (IDP) regions. IDPs have a propensity to fold and form stable structures when bound to an appropriate protein or DNA. The overall goal of this research is to define mechanisms and structural basis for how protein induced folding of the NTD and CTE regulates the activities of these PR domains. This proposal will use combined approaches of molecular biology, biochemistry and protein structure analysis. AIM #1 will define the functional role of two coregulatory proteins that interact with the CTE; HMGB and a bZIP protein termed JDP-2. Mutations in these PR CTE-protein interaction surfaces (identified in AIM#2) and deletion of the hmbg-1 gene will be analyzed for effects on receptor binding with and regulation of endogenous target genes as detected by chromatin immunoprecipitation (ChIP) coupled with microarray (chip), and high through-put qPCR. AIM #2 will use NMR to define interaction sites and structural changes in the PR CTE in response to binding JDP-2 and HMGB and this data will be used to guide functional mutagenesis studies in Aim 1. To further explore the role of the CTE in binding to weak non-canonical DNA sites, NMR structure of PR DBD-CTE complexed with a PRE half-site will be determined. AIM #3 will identify novel PR NTD interacting proteins that induce folding and modulate transcriptional activity of AF-1 and then use these proteins to explore the hypothesis that the NTD has multiple foldable regions and the potential to adopt different functional conformations dependent on the protein partner. This information will be essential for future structure determinations of stable folded NTD-coregulatory protein complexes. PR is required for development, differentiation and maintenance of female reproductive tissues and has a role in reproductive disorders and gynecological cancers including breast cancer. Consequently, PR is an important therapeutic target and small molecule selective PR modulators (SPRMS) are widely used and continue to be developed for a variety of clinical applications. The insights gained from this research on structural properties and molecular mechanisms of the NTD and CTE domains of PR are anticipated to be essential for the development of additional and improved SPRMS. PUBLIC HEALTH RELEVANCE: Progesterone and the progesterone receptor (PR) are essential for development, differentiation and maintenance of female reproductive tissues and have a role in reproductive disorders and gynecological cancers including breast cancer. Consequently, small molecule selective PR modulators (SPRMS) are widely used and continue to be developed for a variety of clinical applications. The research in this application will provide much needed insight into poorly understood structural and functional properties of PR that is essential for the development of additional and improved SPRMS.